Hi,
I am in need of some advise. I am currently running a gradient method using mobile phase A : 250 Mm Amm. Formate and mobile phase B: 100%ACN. My column is a Glycosep N (250x4.6mm).I am running the method on a Waters 2695 and 2475 Flouresence detector (With Empower Software), my expected sample chromatogram contains 9 peaks of intrest. Sample chromatograms achieved are perfect but my method states that a water injection after a sample should be free of contamination.The blank water (HPLC Grade) contains the definate profile of my sample at a % carryover of ~0.15. I have tried flushing the system and have tried different gains and attenuation settings on the detector. Does anyone have any advise on where to go next?
Thanks.

The Waters 2695 system has an needle wash system that must be filled with an appropriate needle wash to wash the needle after the injection. This system must be kept primed and the needle wash solution needs to be kept fresh.
Try using a needle wash that matches your gradient maximum but without the buffer salts added to see if that resolves the problem.
Another thing to check is the condition of the injection system seal pack in the 2695. The seal pack has a lifetime use of around 10000 injections depending on use (high buffer mobile phases tend to wear this part out faster) and this can cause greater carry over issues when it nearing the end of its life. When it begins to leak that is when it has 100% failed.
Hope this helps.
Charlie

Hi Charlie,
Thanks for you suggestions but we change the needlewash daily and prime the needed as part of daily maintenance. I am using a needle wash that has been used before in an originating lab ie. 20% meoh where carryover was not found. Since November, I tried a Glyko N guard column .... but system was overpressuring so have ordered in some differnt guard columns which are used in another site where this method is run. Also, since November I put an new column on .... carryover is gone but am unsure now if column lots are varying??
Have checked that same packing was used by manufacturer and it was........
Will up date you if we solve it!

Rather than a water blank try a maximum gradient composition blank to see if carry over sample peak is still evident or has increased in size. Have you tried the analysis on a different HPLC system with the same column. You need to determine whether the carryover problem is a technical issue with the HPLC or a internal chemistry issue with the column being used etc.
These type of issues are difficult to pin down and time consuming and you are best to use at least two HPLC in good condition to eliminate contamination issues within a particular injection system.

I'd be interested to hear the cause of this issue when you have done more work on it.

Thanks

Charlie

Hi Charlie,
I will definately let you know of any updates, thanks for you suggestions.

G'day from WA

I have a few suggestions also.

1. Is the fluorometer detector being used currently the same as previous (ie same sensitive)?

2. Perhaps the 20% MeOH needle wash was not effective if a lower sensitive detector used and thus carry-over not observed.

3. we use 60% methanol as needle wash for organic acides in soil extracts

4. has the frit at the start of the column been cleaned(?), which will be the case when using a "new" column.

Some thoughts at least!!

Regards
Greg

One can imagine that a sudden disequilibrium can give rise to peaks of analyte hung up on the column, but in the pattern of samples?? If such hung up analytes are slowly washed out of the column one can imagine that an injection of H2O causes negative peaks.
It seems to me that mechanically moving parts (or anything else which is discontinuous) must be involved in the decscribed carryover. Am I ignoring something?

You need to be much more agressive with you needle wash (as was previously mentioned). When needed, with the 2695, a mixture of ACN/IPA/H2O (5:1:1) has shown to be a very effective needle wash. You do not need to make it fresh every day, make a liter and use it untill it is gone. Second thing, before you go any further, change the needle wash frit, it is easy to do (no tools). This frit may be very contaminated at this point and need to be changed. If you keep the needle wash frit clean it is seldom a problem, however once it becomes contaminated, it can be tough to clean.

You need to check to see if the carryover is from the column or needle first. Run a blank injection (-1) w/mobile phase only to see if its the needle then shoot a blank matrix sample to see if its the column. Before each of these injections run a ULOQ.
1. I would try raising the needle height on the injector and increasing your wash vol.
2. I also always try and run an isocratic method first if there's more than 25% carryover from ULOQ into my blank.
3. If my chrom looks bad using an iso method I step up the % starting organic of my mobile phase ie from 10% to 30%.
4. For my injector wash I really like 20:80 MeOH:H20 w/1% HOAC. Works like a champ.
Just a few ideas that have worked for me.
Dalton